The present invention relates to novel proteins having a cytidine deaminase activity; DNAs and fragments thereof (cDNAs, genomic DNAs, and primer DNAs) encoding the proteins; expression vectors comprising the DNAs; transformants transformed with the expression vectors; antibodies reactive to the proteins or fragments thereof; cells producing the antibodies; and methods for identifying substances that regulate production of the proteins, transcription of genes encoding the proteins into mRNAs, or enzyme activities of the proteins.
The germinal center of mammals comprises a highly specialized microenvironment required for the final process of maturation towards antigen specific memory cells and long-lived plasma cells (Embo J., 16:2996-3006, 199; Semin. Immunol., 4:11-17, 1992). In this microenvironment, two fundamental editings of the immunoglobulin genes take place (J. Exp. Med., 173:1165-1175, 1991; Embo. J., 12:4955-4967, 1993; Adv. Exp. Med. Biol., 186:145-151, 1985; Nature, 342:929-931, 1989; Cell, 67:1121-1129).
The first fundamental editing is somatic hypermutation (Curr. Opin. Immunol., 7:248-254, 1995; Annu. Rev. Immunol., 14:441-457, 1996; Science, 244:1152-1157, 1989), a phenomenon in which extensive point mutation in the exons of genes encoding variable regions of immunoglobulins occurs. Accumulation of point mutations leads to selection of B cells expressing high affinity immunoglobulins on their cell surface, accompanied by the affinity maturation of antibodies (Embo. J., 4:345-350, 1985; Proc. Natl. Acad. Sci. USA, 85:8206-8210, 1988). As a result, immunoglobulin genes are edited as new functional genes.
Another fundamental editing process is the class switch recombination (CSR). In CSR, effector functions of antibodies, such as complement fixation, are selected by exchanging exons encoding constant regions of immunoglobulin heavy chains (Curr. Top. Microbiol. Immunol., 217:151-169, 1996; Annu. Rev. Immunol., 8:717-735, 1990).
These two types of genetic editing are very important for effective humoral immunoreaction to eliminate harmful microbes. The molecular mechanisms of the genetic phenomena have not yet been elucidated despite extensive study for several decades.
The present inventors isolated a mouse B cell clone, CH12F3-2, as a research tool to elucidate the molecular mechanism of class switch recombination of immunoglobulin. In this B cell line, class switch recombination (CSR) from IgM to IgA begins several hours after stimulation with IL-4, TGF-xcex2, and CD40L; ultimately, over 80% of the cells become IgA positive (Immunity, 9:1-10, 1998; Curr. Biol., 8:227-230, 1998; Int. Immunol., 8:193-201, 1996).
Using the mouse B cell clone CH12F3-2, the present inventors previously reported that the breakpoints of CSR distribute not only in the switch region (or xe2x80x9cS regionxe2x80x9d), characterized by repeated sequences, but also in neighboring sequences (Curr. Biol., 8:227-230, 1998). However, the breakpoints were rarely seen in I exon and C exon, which are located upstream and downstream of the S region, respectively. Also, according to accumulated scientific evidence, it has been shown that transcription of I exon and C exon and splicing of the transcripts are essential for CSR (Cell, 73:1155-1164, 1993; Science, 259:984-987, 1993; Proc. Natl. Acad. Sci, USA, 90:3705-3709, 1993; Cell, 81:833-836, 1995).
This suggests that the transcripts are involved in CSR either directly or indirectly. Accordingly, the present inventors propose a theory that class switch is initiated by the recognition of DNA-RNA complex structure and not by the recognition of nucleotide sequences of the switch region. This idea is further fortified by the fact that even when the Sa region is substituted with an Sxcex1 region or an Sxcex3 region by introducing a mini-chromosome into the above-mentioned mouse B cell clone CH12P3-2, CSR in the mini-chromosome efficiently occurs after stimulation with cytokines (Immunity, 9:1-10, 1998).
In plants and protozoa, RNA editing, another type of genetic editing, is widely used as a mean for producing functional genes from a limited genome (Cell, 81:833-836, 1995; Cell, 81:837-840, 1995). mRNA editing of many molecules such as the mRNA for apolipoprotein B (apoB), AMPA receptors, Wilmstumor-1, xcex1-galactosidase and neurofibromatosis type-1, and tRNA-Asp, have been reported (Trends Genet., 12:418-424, 1996; Curr. Opin. Genet. Dev., 6:221-231, 1996). Although the molecular mechanism of mammalian RNA editing has not yet been elucidated, one performed by APOBEC-1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1) is becoming understood by degrees (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993).
In apoB RNA editing, the first base C (cytosine) of codon CAA, which encodes glutamine, is converted to U (uridine), which alters the codon to UAA. As a result, an in-frame stop codon is made in the apoB mRNA (J. Cell., 81:187-195, 1995; J. Cell., 50:831-840, 1987; Science, 238:363-266, 1987). apoB-48 and apoB-100 are transcripts of edited mRNA and unedited mRNA of apoB, respectively, and these proteins possess totally different physiological functions from each other (J. Biol. Chem., 271:2353-2356, 1996).
In site-specific RNA-editing, auxiliary factors are required (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993). In the absence of auxiliary factors, APOBEC-1 shows only a cytidine deaminase activity, possessing non-specific low affinity to RNA (J. Biol. Chem., 268:20709-20712, 1993; J. Cell., 81:187-195, 1995; J. Biol. Chem., 270:14768-14775, 1995; J. Biol. Chem., 270:14762-14767, 1995). The expression and activity of the auxiliary factors are found not only in organs with apoB mRNA editing, but also in organs with undetectable levels of APOBEC-1 expression, or organs without apoB mRNA editing (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993; Nucleic Acids Res., 22:1874-1879, 1994; Proc Natl. Acad. Sci, USA, 91:8522-8526, 1994; J. Biol. Chem., 269:21725-21734, 1994).
The unexpected expression of the auxiliary factors involved in apoB mRNA editing suggests that the auxiliary factors may be involved in more general cellular functions or other yet unknown RNA editing. Since the possibility exists that CSR and hypermutation, which are involved in genetic editing of immunoglobulin genes, may be accomplished by RNA editing, it would be very interesting to elucidate whether RNA editing takes place or not in the genetic editing of immunoglobulin genes as mentioned above.
The present invention provides AID (Activation-Induced cytidine Deaminase), a novel cytidine deaminase that is structurally related to APOBEC-1, an RNA editing enzyme, and is involved in RNA editing in germinal center B cells, where genetic editing of immunoglobulin genes occur, and DNA encoding the new enzyme.
The present inventors intensively searched for novel genes involved in class switch recombination (CSR), one of the major types of genetic editing of immunoglobulin genes. As a result, by preparing cDNA libraries for the mouse B cell clone CH12F3-2 (in which class switch recombination from IgM to IgA is shown to occur at an extremely high rate upon activation of the cells by stimulation with cytokines), with and without stimulation with cytokines, and performing subtraction cloning using the libraries, the present inventors found genes encoding mouse- and human-derived novel proteins named AID (Activation-Induced cytidine Deaminase), having a structural relationship to APOBEC-1, one of the RNA editing enzymes, and having a cytidine deaminase activity similar to APOBEC-1.
The AID protein in the present invention possesses features described below, and is considered to be a very important RNA-modifying deaminase involved in regulating B cell activation, CSR of immunoglobulin genes, somatic hypermutation, and affinity maturation, which are all involved in genetic editing specific to germinal center function:
(1) The ORF of the cDNA encoding the AID protein comprises 198 amino acids, with a 24 kDa calculated molecular weight (mouse: SEQ ID NO:2, and human: SEQ ID NO:8). The mouse AID protein shows an approximately 28 kDa molecular weight by SDS-PAGE.
(2) The amino acid sequence of the AID protein is 34% and 26% identical to APOBEC-1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1) at the amino acid sequence level, for mouse and human derived proteins, respectively.
(3) The AID protein has a cytidine/deoxycytidine deaminase motif, which is the active center of the deaminase activity and is conserved in amino acid sequences of proteins belonging to the cytosine nucleoside/nucleotide deaminase family.
(4) The cytidine deaminase motif of the AID protein is allied with an RNA editing deaminase subgroup.
(5) The AID protein has a leucine-rich region considered to be important in protein-protein interaction, similar to APOBEC-1. Four leucines in this leucine-rich region of the AID protein are conserved in the leucine-rich region of APOBEC-1 in rabbit, rat, mouse and human.
(6) In the primary structure of the AID protein, all of the amino acid residues reported to be necessary for APOBEC-1 to bind RNA (Phe66, Phe87, His61, Glu63 and Cys93) are conserved.
(7) The AID protein has a pseudoactive site domain in its C terminal for forming homodimers, similar to APOBEC-1 and ECDDA, an E. coli derived cytidine deaminase. There is a possibility that the AID protein forms homodimers, or associates with other auxiliary proteins.
(8) The AID protein shows a concentration-dependent cytidine deaminase activity. The activity can be inhibited dose dependently by tetrahydrouridine (THU), a specific inhibitor of cytidine deaminase. Also, a zinc chelator, 1,10-o-phenanthroline, inhibits the cytidine deaminase activity of the AID protein while 1,7-o-phenanthroline, the inactive isomer, shows a weak inhibition. Thus, the AID protein can considered to be a zinc-dependent cytidine deaminase, as is APOBEC-1.
(9) Strong expression of AID mRNA is seen in lymph nodes (mesenteric and amygdaline). Also, weak expression in spleen is seen.
(10) Expression of AID mRNA is seen in a variety of lymphoid tissues (Peyer""s patches, mesenteric lymph node, axillary lymph node, spleen, and bone marrow). Especially notable expression is seen in peripheral lymphoid organs, such as lymphatic nodes and Peyer""s patches. In contrast, expression in primary lymphoid organs is lower than the peripheral lymphoid organs.
(11) Expression of AID mRNA is at the lower limit of detection without cytokine (IL-4, CD40L, TGF-xcex2) stimulation in mouse B cell clone CH12F3-2, in which the cytokines stimulate class switch from IgM to IgA in the cells. Expression is induced 3 hours after stimulation, and maximal expression is seen after 12 hours, with cytokine stimulation.
(12) AID mRNA expression in mouse B cell clone CH12F3-2 can be induced more strongly when stimulated with all three cytokines, IL-4, CD40L and TGF-xcex2, simultaneously, than with any one of them alone. Also, it can be considered that de novo protein synthesis is necessary for augmentation of AID mRNA expression, as the AID mRNA expression induction by cytokines in mouse B cell clone CH12F3-2 can be inhibited by cycloheximide, a protein synthesis inhibitor.
(13) In in vitro tests, an augmentation of AID mRNA expression can be seen when normal mouse spleen B cells are stimulated with LPS alone, LPS+IL-4, or LPS+TGF-xcex2.
(14) In in vivo tests, when normal mice are immunized with sheep red blood cells (SRBC), a significant augmentation of AID mRNA expression can be seen 5 days after immunization, in which SRBC are known to induce clonal expansion, germinal center formation, and class switch recombination and affinity maturation of immunoglobulin genes.
(15) The in vivo augmentation of AID mRNA expression by SRBC immunization is specifically seen in splenic CD19 positive B cells.
(16) AID mRNA expression in lymphoid organs is specifically seen in the germinal center, enriched with B cells activated by antigen stimulation.
(17) The human AID gene is located at locus 12p13, close to locus 12p13.1, where the APOBEC-1 gene is located.
According to the characteristics described above, the AID protein of the present invention can be considered to have a function of regulating various biological mechanisms required for generation of antigen-specific immunoglobulins (specific antibodies), which eliminate non-self antigens (foreign antigen, self-reacting cells, etc.) that trigger various diseases. The mechanism for generation of immunoglobulin having high specificity to antigens includes germinal center functions such as activation of B cells, class switch recombination of immunoglobulin genes, somatic hypermutation, and affinity maturation. The AID protein of the present invention can be considered to be one of the enzymes that play an important role in the genetic editing occurring in germinal center B cells (e.g. class switch recombination and somatic mutation).
The dysfunction of the AID protein of the present invention can be the cause of humoral immunodeficiency since it induces failure of germinal center B cell function, such as antigen-specific B cell activation, class switch recombination, and somatic mutation. Conversely, the hyperfunction of the AID protein may induce allergy disease or autoimmune disease since it can cause inappropriate B cell activation and needless class switch recombination and somatic mutation.
Therefore, regulation of the function of AID protein and the gene encoding it enables prevention and treatment of various immunodeficiencies, autoimmune diseases, and allergies, which result from, for example, B cell dysfunctions (e.g., IgA deficiency, IgA nephropathy, xcex3 globulinemia, hyper IgM syndrome, etc.) or class switch deficiency of immunoglobulin. Thus, the AID protein and the gene encoding the AID protein can be targets for the development of drugs for therapy of diseases mentioned above.
Examples of diseases whose onset prevention, symptom remission, therapy and/or symptomatic treatment effect is expected by regulating the function of the AID protein of the present invention or the gene encoding it include, for example, primary immunodeficiency syndrome with congenital disorder of immune system, mainly various immunodeficiencies considered to develop by B cell deficiency, decrease, or dysfunction (e.g., sex-linked agammaglobulinemia, sex-linked agammaglobulinemia with growth hormone deficiency, immunoglobulin deficiency with high IgM level, selective IgM deficiency, selective IgE deficiency, immunoglobulin heavy chain gene deletion, xcexa chain deficiency, IgA deficiency, IgG subclass selective deficiency, CVID (common variable immunodeficiency), infantile transient dysgammaglobulinemia, Rosen syndrome, severe combined immunodeficiency (sex-linked, autosomal recessive), ADA (adenosine deaminase) deficiency, PNP (purine nucleoside phosphorylase) deficiency, MHC class II deficiency, reticular dysplasia, Wiskott-Aldrich syndrome, ataxia telangiectasia, DiGeorge syndrome, chromosomal aberration, familial Ig hypermetabolism, hyper IgE syndrome, Gitlin syndrome, Nezelof syndrome, Good syndrome, osteodystrophy, transcobalamin syndrome, secretory bead syndrome, etc.), various diseases with antibody production deficiency that are secondary immunodeficiency syndromes with a disorder of immune system caused by an acquired etiology (for example, AIDS, etc.), and/or various allergic diseases (e.g., bronchial asthma, atopic dermatitis, conjunctivitis, allergic rhinitis, allergic enteritis, drug-induced allergy, food allergy, allergic urticaria, glomerulonephritis, etc.).
The AID proteins of the present invention, a fragment thereof, a DNA encoding the AID protein, a fragment thereof, and an antibody against the AID protein are useful as reagents for developing drugs for prevention and therapy of such diseases.
Also, the DNA itself is useful as an antisense drug regulating the function of the AID gene at a gene level and in gene therapy. The protein or the fragments thereof (e.g. enzyme active site) themselves are useful as drugs.
Furthermore, a DNA comprising a nucleotide sequence that is complementary to an arbitrary partial nucleotide sequence in the nucleotide sequence of genomic DNA encoding AID protein of the present invention (especially human AID protein) is useful as a primer DNA for polymerase chain reaction (PCR).
An arbitrary partial nucleotide sequence of genomic DNA encoding the AID protein (especially human AID protein) of the present invention can be amplified by PCR using the primer DNA pair. For example, in the case that mutation or deletion of the nucleotide sequence of genomic DNA (especially exon) encoding AID protein is presumed to cause a certain immunodeficiency or an allergy, mutations and deletions in the genomic DNA can be identified by amplifying an arbitrary partial nucleotide sequence of genomic DNA encoding the AID protein obtained from tissue or cells of immunodeficiency or allergy patients by PCR using a pair of primer DNAs, by analyzing the presence and the size of PCR products and the nucleotide sequence of the PCR products, and by comparing the nucleotide sequence with the corresponding nucleotide sequence in the genomic DNA encoding the AID protein derived from a normal human. That is to say, this method is capable of not only, for example, elucidating relationships between immunodeficiency or allergy and AID protein, but also, in the case where the AID protein is the cause of onset of a sort of disease (e.g. immunodeficiency and/or allergy), diagnosing the disease by the methods mentioned above.
Furthermore, an antibody reactive to the AID protein of the present invention or a fragment thereof is extremely useful as an antibody drug by regulating functions of the AID protein.
Furthermore, the gene (DNA), protein, and antibody of the present invention are useful as reagents for searching for substrates (e.g. RNA, etc.) that interact (binding) with the protein (enzyme) of the present invention, or other auxiliary proteins associated with the protein of the present invention, and for developing drugs targeting the substrates and auxiliary proteins.
Also, model animals can be generated by disrupting (inactivating) the AID gene based on the genetic information on the AID protein derived from mammals (e.g. mouse, etc.), which is one embodiment of the DNA of present invention. By analyzing the physical, biological, pathological, and genetic features of the model animal, it is possible to elucidate functions of the genes and the proteins of the present invention.
Furthermore, by introducing a normal human AID gene or mutant human AID gene (e.g. mutant human AID genes derived from immunodeficiency patients), which is one embodiment of the present invention, into the model animal whose endogenous gene has been disrupted, model animals having only normal or mutant human AID genes of the present invention can be generated. By administering drugs (compounds, antibodies, etc.) targeting the introduced human AID genes to the model animals, therapeutic effects of the drugs can be evaluated.
Furthermore, a method for identifying a substance that regulates production of the AID protein of the present invention or transcription of a gene encoding the AID protein into mRNA, or a substrate that inhibits the enzyme activity of the AID protein (e.g. cytidine deaminase activity) is extremely useful as a means to develop drugs for therapy and prevention of various diseases (especially, immunodeficiency and/or allergy) in which the above-mentioned AID protein or AID gene is considered to be involved.
Thus, the present invention, for the first time, provides the below-mentioned DNAs (cDNAs, genomic DNAs, and an arbitrary fragment thereof), proteins, expression vectors, transformants, antibody pharmaceutical compositions, cells, the use of the DNA fragments as primer DNAs, and methods for screening.
(1) A DNA or a fragment thereof encoding a protein comprising the amino acid sequence of SEQ ID NO:2 or 8.
(2) The DNA or the fragment of (1), wherein the protein has a cytidine deaminase activity.
(3) A DNA or a fragment thereof comprising the nucleotide sequence of SEQ ID NO:1 or 7.
(4) A DNA or a fragment thereof comprising a nucleotide sequence of (a) or (b) below:
(a) a nucleotide sequence comprising the nucleotide residues 93 to 689 of SEQ ID NO:1 or
(b) a nucleotide sequence comprising the nucleotide residues 80 to 676 of SEQ ID NO:7.
(5) A DNA or a fragment thereof of (a) or (b) below:
(a) a DNA or a fragment thereof that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:2 and having a cytidine deaminase activity or
(b) a DNA or a fragment thereof that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:8 and having a cytidine deaminase activity.
(6) A protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO:2 or 8.
(7) A protein or a fragment thereof comprising substantially the same amino acid sequence as that of SEQ ID NO:2 or 8 and having a cytidine deaminase activity.
(8) A protein of (a) or (b) below.
(a) a mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:2, and that has a cytidine deaminase activity, or
(b) a mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:8, and that has a cytidine deaminase activity.
(9) An expression vector comprising the DNA or the fragment of any one of (1) to (5).
(10) A transformant transformed with the expression vector of (9).
(11) An antibody or a portion thereof reactive to the protein of any one of (6) to (8) or to a fragment of the protein.
(12) The antibody or the portion thereof of (11), wherein the antibody is a monoclonal antibody.
(13) A pharmaceutical composition comprising the antibody or the portion thereof of (11) or (12), and a pharmaceutically acceptable carrier.
(14) A cell producing a monoclonal antibody reactive to the protein of any one of (6) to (8) or to a fragment of the protein.
(15) The cell of (14), wherein the cell is a hybridoma obtained by fusing, with a mammal-derived myeloma cell, a non-human mammal-derived B cell that produces a monoclonal antibody.
(16) The cell of (15), wherein the cell is a transgenic cell transformed by introducing, into a cell, either or both of a DNA encoding a heavy chain of the monoclonal antibody and a DNA encoding a light chain of the monoclonal antibody.
(17) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (c) below:
(a) SEQ ID NO:9,
(b) SEQ ID NO:10, or
(c) SEQ ID NO:35.
(18) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (e) below:
(a) SEQ ID NO:11,
(b) SEQ ID NO:12,
(c) SEQ ID NO:13,
(d) SEQ ID NO:14, or
(e) SEQ ID NO:15.
(19) A DNA comprising a nucleotide sequence complementary to an arbitrary partial nucleotide sequence of a nucleotide sequence of any one of (a) to (h) below:
(a) SEQ ID NO:9,
(b) SEQ ID NO:10,
(c) SEQ ID NO:11,
(d) SEQ ID NO:12,
(e) SEQ ID NO:13,
(f) SEQ ID NO:14,
(g) SEQ ID NO:15, or
(h) SEQ ID NO:25.
(20) The DNA of (19), wherein the DNA comprises a nucleotide sequence of any one of (a) to (q) below:
(a) SEQ ID NO:18,
(b) SEQ ID NO:19,
(c) SEQ ID NO:20,
(d) SEQ ID NO:21,
(e) SEQ ID NO:22,
(f) SEQ ID NO:23,
(g) SEQ ID NO:24,
(h) SEQ ID NO:25,
(i) SEQ ID NO:26,
(j) SEQ ID NO:27,
(k) SEQ ID NO:28,
(l) SEQ ID NO:29,
(m) SEQ ID NO:30,
(n) SEQ ID NO:31,
(o) SEQ ID NO:32,
(p) SEQ ID NO:33, or
(q) SEQ D NO:34.
(21) Use of the DNA of (19) or (20) as a primer DNA in polymerase chain reaction.
(22) Use of a pair of DNAs of any one of (a) to (n) below as primer DNAs in polymerase chain reaction:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO:31 and a DNA comprising the nucleotide sequence of SEQ ID NO:32,
(b) a DNA comprising the nucleotide sequence of SEQ ID NO:20 and a DNA comprising the nucleotide sequence of SEQ ID NO:22,
(c) a DNA comprising the nucleotide sequence of SEQ ID NO:21 and a DNA comprising the nucleotide sequence of SEQ ID NO:30,
(d) a DNA comprising the nucleotide sequence of SEQ ID NO:24 and a DNA comprising the nucleotide sequence of SEQ ID NO:25,
(e) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,
(f) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,
(g) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,
(h) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,
(i) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,
(g) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,
(k) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,
(l) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,
(m) a DNA comprising the nucleotide sequence of SEQ ID NO:33 and a DNA comprising the nucleotide sequence of SEQ ID NO:29, or,
(n) a DNA comprising the nucleotide sequence of SEQ ID NO:18 and a DNA comprising the nucleotide sequence of SEQ ID NO:19.
(23) A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of:
(a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein and
(b) (i) comparing the level of the AID protein produced by the cells cultured in the presence of the substance with the level of the AID protein produced by the cells cultured in the absence of the substance or
(ii) comparing the level of the AID protein-encoding mRNA transcribed in the cells cultured in the presence of the substance with the level of the AID protein-encoding mRNA transcribed in the cells cultured in the absence or the substance.
(24) A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of:
(a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein and a protein other than the AID protein, wherein transcription of a gene encoding the other protein into mRNA is dependent in the cells on the degree of a signal of transcription of the gene encoding the AID protein into mRNA and
(b) comparing the level of the other protein produced by the cells cultured in the presence of the substance with the level of the other protein produced by the cells cultured in the absence of the substance.
(25) The method of (23) or (24), wherein the cells are transgenic cells transformed with a gene encoding the protein.
(26) The method of (24), wherein the cells are transgenic cells transformed with a gene encoding the protein and a gene encoding the other protein.
(27) The method of (26), wherein the protein is a reporter protein.
(28) The method of (27), wherein comparison of the level of the other protein is comparison of the level of a signal generated by the reporter protein.
(29) The method of (27) or (28), wherein the reporter protein is luciferase.
(30) A method for identifying a substance that inhibits an enzyme activity of an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8, the method comprising the step of (a) or (b) below:
(a) culturing, separately in the presence and the absence of the substance, mammal-derived B cells or tissues comprising the B cells, and comparing enzyme activities of the AID protein in the B cells separately cultured or
(b) (i) administering the substance separately to an AID gene knockout mouse whose endogenous AID gene is inactivated so that transcription of the endogenous AID gene into mRNA is inhibited, and to a normal mouse and
(ii) comparing enzyme activities of the AID proteins in the B cells isolated from the respective mice.
(31) The method of (30), wherein the enzyme activity is a cytidine deaminase activity.
Hereafter, the present invention is explained in detail, by clarifying the terms used in the present invention and general methods for producing the proteins, DNAs, antibodies, and cells of the present invention.
The xe2x80x9cprotein or a fragment thereofxe2x80x9d means a protein and a fragment thereof derived from a mammal such as human, bovine, sheep, pig, goat, rabbit, rat, hamster, guinea pig, mouse, and so on, preferably a protein or a fragment thereof derived from human, rabbit, rat, or mouse, and particularly preferably, a protein or a fragment thereof derived from human or mouse.
As a particularly preferred embodiment, it means any protein or a fragment thereof below.
(1) A protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO:2 or 8.
(2) A protein or a fragment thereof comprising substantially the same amino acid sequence as that of SEQ D NO:2 or 8 and having a cytidine deaminase activity.
(3) A mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:2, and that has a cytidine deaminase activity.
(4) A mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:8, and that has a cytidine deaminase activity.
Here, xe2x80x9chaving substantially the same amino acid sequencexe2x80x9d means that a protein has an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in the amino acid sequence shown in the references are substituted, deleted, and/or modified, and that a protein has an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, are added to the amino acid sequence shown in the references.
The protein of the present invention includes monomer molecules, homodimers in which one strand binds to another strand comprising an identical amino acid sequence, heterodimers in which one strand binds to another strand comprising a different amino acid sequence, and oligomers such as trimers or tetramers.
Also, a xe2x80x9cfragment of a proteinxe2x80x9d means an arbitrary partial sequence (fragment) in the amino acid sequence that the above-mentioned AID protein of the present invention comprises. For example, it includes an enzyme active site required for the AID protein to exert an enzyme activity represented by a cytidine deaminase activity, and an interaction site required for the AID protein to bind or associate with substrates (e.g. mRNA, etc.) or various auxiliary proteins.
Alphabetical triplet or single letter codes used to represent amino acids in the present specification or figures mean amino acids as follows:
(Gly/G), glycine; (Ala/A), alanine; (Val/V), valine; (Leu/L), leucine; (Ile/I), isoleucine; (Ser/S), serine; (Thr/T), threonine; (Asp/D), aspartic acid; (Glu/E), glutamic acid; (Asn/N), asparagines; (Gln/Q) glutamine; (Lys/K), lysine; (Arg/R), arginine; (Cys/C), cysteine; (Met/M), methionine; (Phe/F), phenylalanine; (Tyr/Y), tyrosine; (Trp/W), tryptophan; (His/H), histidine; (Pro/P), proline.
The proteins and fragments of the present invention can be produced by properly using, in addition to genetic engineering technique mentioned below, methods well known in the art, such as chemical synthesis, cell culture method, and so on, or their modified methods.
Also, the AID protein of the present invention can be produced as a recombinant fusion protein with another protein (e.g. GST (Glutathione S-transferase), etc.). In this case, the fusion protein is advantageous in that it can be extremely easily purified by affinity chromatography employing adsorbent on which another molecule binding specifically to GST is immobilized. Moreover, since various antibodies reactive to GST are provided, the quantification of the fusion protein can be simply carried out by immunoassay (e.g. ELISA, etc.) using antibodies against GST.
The DNA of the present invention is a DNA encoding a protein of the present invention and a fragment thereof, and it includes any nucleotide sequence encoding the protein of the present invention and includes both genomic DNAs and cDNAs. Also, the DNA includes any DNA composed of any codons as long as the codons encode identical amino acids.
Also, the DNA of the present invention includes a DNA encoding a mammalian AID protein, and, as a preferred embodiment, a DNA encoding a mouse AID protein or a human AID protein can be exemplified.
Examples of specific embodiments are as follows:
(1) A DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:2 or 8.
(2) The DNA of (1), wherein the protein has a cytidine deaminase activity.
(3) A DNA comprising the nucleotide sequences of SEQ ID NO:1 or 7.
(4) A DNA comprising nucleotides s 93 to 689 of SEQ ID NO:1.
(5) A DNA comprising nucleotides 80 to 676 of SEQ ID NO:7.
(6) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:2 and having a cytidine deaminase activity.
(7) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:8 and having a cytidine deaminase activity.
(8) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (c) below:
(a) SEQ ID NO:9,
(b) SEQ ID NO:10, or
(c) SEQ ID NO:35.
(9) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (e) below:
(a) SEQ ID NO:11,
(b) SEQ ID NO:12,
(c) SEQ ID NO:13,
(d) SEQ ID NO:14, or
(e) SEQ ID NO:15.
(10) A DNA comprising a complementary nucleotide sequence to an arbitrary partial sequence of a nucleotide sequence of any one of (a) to (h) below:
(a) SEQ ID NO:9,
(b) SEQ ID NO:10,
(c) SEQ ID NO:11,
(d) SEQ ID NO:12,
(e) SEQ ID NO:13,
(f) SEQ ID NO:14,
(g) SEQ ID NO:15, or
(h) SEQ ID NO:35.
(11) A DNA comprising a nucleotide sequence of any one of (a) to (q) below:
(a) SEQ ID NO:18,
(b) SEQ ID NO:19,
(c) SEQ ID NO:20,
(d) SEQ ID NO:21,
(e) SEQ ID NO:22,
(f) SEQ ID NO:23,
(g) SEQ ID NO:24,
(h) SEQ ID NO:25,
(i) SEQ ID NO:26,
(j) SEQ ID NO:27,
(k) SEQ ID NO:28,
(l) SEQ ID NO:29,
(m) SEQ ID NO:30,
(n) SEQ ID NO:31,
(o) SEQ ID NO:32,
(p) SEQ ID NO:33, or,
(q) SEQ ID NO:34.
Furthermore, a DNA encoding a mutant protein or a fragment thereof obtained by substituting, deleting, and/or modifying multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, or by inserting multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids in the amino acid sequence constituting the above-defined AID protein of the present invention or a fragment thereof is included in the DNA of the present invention.
The term xe2x80x9cunder stringent conditionsxe2x80x9d used herein means, for example, the following conditions. For example, in the case of carrying out hybridization using a probe with not less than 50 bases in 0.9% NaCl, target temperature of causing 50% dissociation (Tm) can be calculated from the formula below, and the hybridization temperature can be set as the formula below.
Tm=82.3xc2x0 C.+0.41xe2x80x2(G+C)%xe2x88x92500/nxe2x88x920.61xc3x97(formamide)%
(n means the number of bases of the probe)
Temperature=Tmxe2x88x9225xc2x0 C.
Also, in the case of using a probe with not less than 100 bases (G+C=40 to 50%), the changes of Tm as (1) and (2) below can be used as the indicator.
(1) Every 1% mismatch decreases Tm by approximately 1xc2x0 C.
(2) Every 1% formamide decreases Tm by 0.6 to 0.7xc2x0 C.
Thus, the temperature condition in the case of combination of complete complementary strands can be set as below.
(A) 65 to 75xc2x0 C. (without formamide)
(B) 35 to 45xc2x0 C. (with 50% formamide)
The temperature condition in the case of combination of incomplete complementary strands can be set as below.
(A) 45 to 55xc2x0 C. (without formamide)
(B) 35 to 42xc2x0 C. (with 30% formamide)
In the case of using probes with not more than 23 bases, temperature can be 37xc2x0 C., or the formula below can also be used as an indicator.
Temperature=2xc2x0 C.xc3x97(number of A+T)+4xc2x0 C.xc3x97(number of C+G)xe2x88x925xc2x0 C.
The DNA of the present invention can be a DNA obtained by any method. For example, the DNA includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCR amplification with RNA or DNA as a template, and DNA constructed by appropriately combining these methods.
As used herein, an xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in random, uncharacterized mixtures of different DNA molecules, transfected cells, or cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. For example, the substantially pure polypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight. Purity can be measured by any appropriate standard method known in the art, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The invention also includes a polypeptide, or fragment thereof, that differs from the corresponding sequence shown as SEQ ID NO:2 or 8. The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In one embodiment, the polypeptide includes an amino acid sequence at least about 60% identical to a sequence shown as SEQ ID NO:2 or 8, or a fragment thereof. Preferably, the polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:2 or 8 and has at least one cytidine deaminase function or activity described herein. Preferred polypeptide fragments of the invention are at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of the sequence shown as SEQ ID NO:2 or 8 and have at least one cytidine deaminase activity described herein. Or alternatively, the fragment can be merely an immunogenic fragment.
As used herein, xe2x80x9c% identityxe2x80x9d of two amino acid sequences, or of two nucleic acid sequences, is determined using the algorithm of Karlin and Altschul (PNAS USA 87:2264-2268, 1990), modified as in Karlin and Altschul, PNAS USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignment for comparison purposes GappedBLAST is utilized as described in Altschul et al (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention.
Accordingly, in one aspect, the invention provides an isolated or purified nucleic acid molecule that encodes a polypeptide described herein or a fragment thereof Preferably, the isolated nucleic acid molecule includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO:1 or 7. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequence shown in SEQ ID NO:1 or 7. In the case of an isolated nucleic acid molecule which is longer than or equivalent in length to the reference sequence, e.g., SEQ ID NO:1 or 7, the comparison is made with the full length of the reference sequence. Where the isolated nucleic acid molecule is shorter that the reference sequence, e.g., shorter than SEQ ID NO:1 or 7, the comparison is made to a segment of the reference sequence of the same length (excluding any loop required by the homology calculation).
The DNA encoding the protein of the present invention can be prepared by the usual methods: cloning cDNA from mRNA encoding the protein of the present invention, isolating genomic DNA and splicing it, chemical synthesis, and so on.
(1) cDNA can be cloned from mRNA encoding the protein of the present invention by, for example, the method described below.
First, the mRNA encoding the protein of the present invention is prepared from the above-mentioned tissues or cells expressing and producing the protein of the present invention. mRNA can be prepared by isolating total RNA by a known method such as guanidine-thiocyanate method (Chirgwin et al., Biochemistry, 18:5294, 1979), hot phenol method, or AGPC method, and subjecting it to affinity chromatography using oligo-dT cellulose or poly-U Sepharose.
Then, with the mRNA obtained as a template, cDNA is synthesized, for example, by a well-known method using reverse transcriptase, such as the method of Okayama et al (Mol. Cell. Biol. 2:161 (1982); Mol. Cell. Biol. 3:280 (1983)) or the method of Hoffman et al. (Gene 25:263 (1983)), and converted into double-stranded cDNA. A cDNA library is prepared by transforming E. coli with plasmid vectors, phage vectors, or cosmid vectors having this cDNA or by transfecting E. coli after in vitro packaging.
The plasmid vectors used in this invention are not limited as long as they are replicated and maintained in hosts. Any phage vector that can be replicated in hosts can also be used. Examples of usually used cloning vectors are pUC19, xcexgt10, xcexgt11, and so on. When the vector is applied to immunological screening as mentioned below, a vector having a promoter that can express a gene encoding the desired protein in a host is preferably used.
cDNA can be inserted into a plasmid by, for example, the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can be inserted into a phage vector by, for example, the method of Hyunh et al. (DNA cloning, a practical approach, 1, p.49 (1985)). These methods can be simply performed by using a commercially available cloning kit (for example, a product from Takara Shuzo). The recombinant plasmid or phage vector thus obtained is introduced into an appropriate host cell such as a prokaryote (for example, E. coli: HB101, DH5a, MC1061/P3, etc).
Examples of a method for introducing a plasmid into a host are, calcium chloride method, calcium chloride/rubidium chloride method and electroporation method, described in Molecular Cloning, A Laboratory Manual (second edition, Cold Spring Harbor Laboratory, p.1.74 (1989)). Phage vectors can be introduced into host cells by, for example, a method in which the phage DNAs are introduced into grown hosts after in vitro packaging. In vitro packaging can be easily performed with a commercially available in vitro packaging kit (for example, a product from Stratagene or Amersham).
The identification of cDNA encoding protein, its expression being augmented depending on the stimulation of cytokines like AID protein of the present invention, can be carried out by for example suppression subtractive hybridization (SSH)(Proc. Natl. Acad. Sci. USA, 93:6025-6030, 1996; Anal. Biochem., 240:90-97, 1996) taking advantage of suppressive PCR effect (Nucleic Acids Res., 23:1087-1088, 1995), using two cDNA libraries, namely, a cDNA library constructed from mRNA derived from stimulated cells (tester cDNA library) and one constructed from mRNA derived from unstimulated cells (driver cDNA library).
The preparation of cDNA libraries required for subtraction cloning can be performed by using a commercially available kit, for example, PCR-Select Subtraction Kit (CLONTECH, cat: K1804-1). The experiment can be performed according to the instructions accompanying the kit.
An example of a practical experimental procedure is listed below, briefly.
PolyA+ RNA is prepared from cells with or without stimulation with appropriate stimulant as previously reported (Nucleic Acids Res., 26:911-918, 1998). Next, cDNA is prepared, using reverse transcriptase, from each polyA+ RNA sample, as is the commonly used method. cDNA prepared from stimulated cells is used as tester cDNA and that prepared from unstimulated cells as driver cDNA.
According to the previous report mentioned above and experimental manuals accompanying the kit, driver cDNA is added to tester cDNA to perform subtraction. The efficiency of subtraction is monitored by adding small amount of exogenous DNA as a control. After subtraction, the exogenous DNA is concentrated.
The subtracted cDNA is cloned into an appropriate plasmid expression vector to construct a plasmid library by a commonly used method.
Similar to the previously reported method, many colonies are screened by differential hybridization method (Nucleic Acids Res., 26:911-918, 1998; RINSYO-MEN-EKI, 29:451-459, 1997). Here, as the hybridization probes, tester cDNA and driver cDNA mentioned above labeled with radioisotope can be used. Clones containing the objective DNA or containing exogenous DNA can be distinguished by hybridizing the exogenous DNA with replicant filters.
Objective cDNA or its fragment can be obtained by selecting clones giving strong signals against radiolabeled tester cDNA probe rather than radiolabeled driver cDNA probe.
Also, cDNA encoding the protein of the present invention can be accomplished by other general cDNA screening methods.
For instance, cDNA or a fragment encoding the protein of the present invention cloned by subtraction cloning method mentioned above, or chemically synthesized oligonucleotides corresponding to an amino acid sequence of the protein of the present invention, are labeled with 32P to make probes, then by well-known colony hybridization methods (Crunstein et al., Proc. Natl. Acid. Sci. USA, 72:3961, 1975) or plaque hybridization methods (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.2.108, 1989), commercial or originally prepared cDNA libraries can be screened. Furthermore, a method to amplify DNA including cDNA encoding the protein of the present invention by PCR, by constructing a pair of PCR primers based on cDNA or its fragment encoding the protein of the present invention isolated by the subtraction cloning mentioned above, can be listed.
When a cDNA library prepared using a cDNA expression vector is used, the desired clone can be screened by the antigen-antibody reaction using an antibody against the desired protein. A screening method using PCR methodology is preferably used when many clones are subjected to screening.
The nucleotide sequence of the DNA thus obtained can be determined by the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA, 74:560 (1977)) or the dideoxynucleotide synthetic chain termination method using phage M13 (Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977)). The nucleotide sequence can be easily determined using a commercial DNA sequencer.
The whole or a part of the gene encoding the protein of the present invention can be obtained by excising the clone obtained as mentioned above with restriction enzymes and so on.
(2) Also, the DNA encoding the protein of the present invention can be isolated from genomic DNA derived from the cells expressing the protein of the present invention as mentioned above by the following methods.
Such cells are solubilized preferably by SDS or proteinase K, and the DNAs are deproteinized by repeating phenol extraction. RNAs are digested preferably with ribonuclease. The DNAs obtained are partially digested with appropriate restriction enzymes, and the DNA fragments obtained are amplified with appropriate phage or cosmid to generate a library. Then, clones having the desired sequence are detected, for example, by using radioactively labeled DNA probes, and the whole or a portion of the gene encoding the protein of the present invention is obtained from the clones by excision with restriction enzymes, etc.
For example, cDNA encoding a human-derived protein can be obtained by preparing a cosmid library into which human genomic DNAs (chromosomal DNAs) are introduced (xe2x80x9cLaboratory Manual Human Genome Mapping,xe2x80x9d M. Hori and Y. Nakamura, eds., Maruzen), screening the cosmid library to obtain positive clones containing DNA corresponding to the coding region of the desired protein, and screening the above cDNA library using the coding region DNA excised from the positive clones as a probe.
Also, the present invention relates to any fragment of DNA (cDNA, genomic DNA, etc.) encoding an AID protein (especially a human AID protein) of the present invention described above. DNA with a nucleotide sequence complementary to any nucleotide sequence of cDNA or genomic DNA is useful as a primer DNA in polymerase chain reaction (PCR). By PCR using a pair of primer DNAs, any partial nucleotide sequence of genomic DNA encoding AID protein (especially human AID protein) of the present invention can be amplified.
For instance, in the case that mutation or deletion of genomic DNA (especially exon) encoding the AID protein is presumed to cause a certain immunodeficiency or allergy, the existence of such a mutation or deletion can be analyzed by PCR described below.
(1) Prepare a pair of primers comprising nucleotide sequence complementary to any partial nucleotide sequence of genomic DNA encoding an AID protein of the present invention.
(2) Amplify the objective partial nucleotide sequence of the genomic DNA using the pair of primers, using genomic DNA encoding AID protein obtained from tissue or cells of immunodeficiency or allergy patients as templates.
(3) Analyze the existence of PCR products and the nucleotide sequence of the PCR products, and identify the mutation and deletion in the genomic DNA by comparing the nucleotide sequence and corresponding nucleotide sequence of genomic DNA encoding AID protein derived from a normal human.
Thus, the method described above can not only elucidate, for example, the relationship between immunodeficiency and/or allergy and AID protein, but also be used for the diagnosis of a certain kind of disease, in the case that AID protein is the cause of the disease.
Examples of the nucleotide sequence of the primer DNA are as follows:
(1) A DNA comprising a complementary nucleotide sequence to an arbitrary partial sequence of a nucleotide sequence of any one of (a) to (h) below:
(a) SEQ ID NO:9,
(b) SEQ ID NO:10,
(c) SEQ ID NO:11,
(d) SEQ ID NO:12,
(e) SEQ ID NO:13,
(f) SEQ ID NO:14,
(g) SEQ ID NO:15, or
(h) SEQ ID NO:35.
(2) A DNA comprising a nucleotide sequence of any one of (a) to (q) below:
(a) SEQ ID NO:18,
(b) SEQ ID NO:19,
(c) SEQ ID NO:20,
(d) SEQ ID NO:21,
(e) SEQ ID NO:22,
(f) SEQ ID NO:23,
(g) SEQ ID NO:24,
(h) SEQ ID NO:25,
(i) SEQ ID NO:26,
(j) SEQ ID NO:27,
(k) SEQ ID NO:28,
(l) SEQ ID NO:29,
(m) SEQ ID NO:30,
(n) SEQ ID NO:31,
(o) SEQ ID NO:32,
(p) SEQ ID NO:33, or,
(q) SEQ ID NO:34.
Also, the present invention relates to the use of the above-mentioned DNA fragment as a primer DNA in polymerase chain reaction.
Examples of the combination of primer DNAs for PCR in diagnosis accomplished by PCR gene amplification and by analyzing it are as follows:
(1) a DNA comprising the nucleotide sequence of SEQ ID NO:31 and a DNA comprising the nucleotide sequence of SEQ ID NO:32,
(2) a DNA comprising the nucleotide sequence of SEQ ID NO:20 and a DNA comprising the nucleotide sequence of SEQ ID NO:22,
(3) a DNA comprising the nucleotide sequence of SEQ ID NO:21 and a DNA comprising the nucleotide sequence of SEQ ID NO:30,
(4) a DNA comprising the nucleotide sequence of SEQ ID NO:24 and a DNA comprising the nucleotide sequence of SEQ ID NO:25,
(5) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,
(6) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,
(7) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,
(8) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,
(9) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,
(10) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,
(11) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,
(12) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,
(13) a DNA comprising the nucleotide sequence of SEQ ID NO:33 and a DNA comprising the nucleotide sequence of SEQ ID NO:29, or,
(14) a DNA comprising the nucleotide sequence of SEQ ID NO:18 and a DNA comprising the nucleotide sequence of SEQ ID NO:19.
Moreover, the present invention also relates to a recombinant vector comprising the DNA encoding the protein of the present invention. As a recombinant vector of the present invention, any vector can be used as long as it is capable of retaining replication or self-multiplication in each host cell of prokaryotic and/or eukaryotic cells, including plasmid vectors and phage vectors.
The recombinant vector can easily be prepared by ligating the DNA encoding a protein of the present invention with a vector for recombination available in the art (plasmid DNA and bacteriophage DNA) by the usual method.
Specific examples of the vectors used for recombination are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples of phages are a bacteriophage such as xcex phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.
An expression vector is useful for expressing the DNA encoding the protein of the present invention and for producing the protein of the present invention. The expression vector is not limited as long as it expresses the gene encoding the protein of the present invention in various prokaryotic and/or eukaryotic host cells and produces this protein. Examples thereof are pMAL C2, pEF-BOS (Nucleic Acids Res. 18:5322 (1990) and so on), pME18S (Experimental Medicine: SUPPLEMENT, xe2x80x9cHandbook of Genetic Engineeringxe2x80x9d (1992) and so on), etc.
Also, the protein of the present invention can be produced as a fusion protein with other proteins. It can be prepared as a fusion protein, for example, with GST (Glutathione S-transferase) by subcloning a cDNA encoding the protein of the present invention, for example, into plasmid pGEX4T1 (Pharmacia), by transforming E. coli DH5xcex1, and by culturing the transformant.
When bacteria, particularly E. coli, are used as host cells, an expression vector generally comprises, at least, a promoter/operator region, an initiation codon, the DNA encoding the protein of the present invention, termination codon, terminator region, and replicon.
When yeast, animal cells, or insect cells are used as hosts, an expression vector is preferably comprising, at least, a promoter, an initiation codon, the DNA encoding the protein of the present invention, and a termination codon. It may also comprise the DNA encoding a signal peptide, enhancer sequence, 5xe2x80x2- and 3xe2x80x2-untranslated region of the gene encoding the protein of the present invention, splicing junctions, polyadenylation site, selectable marker region, and a replicon. The expression vector may also contain, if required, a gene for gene amplification (marker) that is usually used.
A promoter/operator region to express the protein of the present invention in bacteria comprises a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is Escherichia, it preferably comprises Trp promoter, lac promoter, recA promoter, xcexPL promoter, lpp promoter, tac promoter, or the like. Examples of a promoter to express the protein of the present invention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, and so on. When the host is Bacillus, examples thereof are SL01 promoter, SP02 promoter, penP promoter, and so on. When the host is a eukaryotic cell such as a mammalian cell, examples thereof are SV40-derived promoter, retrovirus promoter, heat shock promoter, and so on, and preferably an SV-40 or retrovirus-derived one. As a matter of course, the promoter is not limited to the above examples. In addition, using an enhancer is effective for expression.
A preferable initiation codon is, for example, a methionine codon (ATG).
A commonly used termination codon (for example, TAG, TAA, TGA) is exemplified as a termination codon.
Usually, natural or synthetic terminators are used as a terminator region.
A replicon means a DNA capable of replicating the whole DNA sequence in host cells, and includes a natural plasmid, an artificially modified plasmid (DNA fragment prepared from a natural plasmid), a synthetic plasmid, and so on. Examples of preferable plasmids are pBR322 or its artificial derivatives (DNA fragment obtained by treating pBR322 with appropriate restriction enzymes) for E. coli, yeast 2xcexc plasmid or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, and such for mammalian cells.
An enhancer sequence, polyadenylation site, and splicing junction that are usually used in the art, such as those derived from SV40, can also be used.
A selectable marker usually employed can be used according to the usual method. Examples thereof are resistance genes for antibiotics, such as tetracycline, ampicillin, or kanamycin.
Examples of genes for gene amplification are dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene, aspartate transcarbamylase gene, etc.
The expression vector of the present invention can be prepared by continuously and circularly linking at least the above-mentioned promoter, initiation codon, DNA encoding the protein of the present invention, termination codon, and terminator region, to an appropriate repticon. If desired, appropriate DNA fragments (for example, linkers, restriction sites, and so on), can be used by the usual method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.
Transformants of the present invention can be prepared by introducing the expression vector mentioned above into host cells.
Host cells used in the present invention are not limited as long as they are compatible with an expression vector mentioned above and can be transformed. Examples thereof are various cells such as wild-type cells or artificially established recombinant cells usually used in the technical field of the present invention (for example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces, Pichia, and such), animal cells, or insect cells).
E. coli or animal cells are preferably used. Specific examples are E. coli (DH5xcex1, TB1, HB101, and such), mouse-derived cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, and such), rat-derived cells (PC12, PC12h), hamster-derived cells (BHK, CHO, and such), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, and such), and human-derived cells (Hela, diploid fibroblast-derived cells, myeloma cells, and HepG2, and such).
An expression vector can be introduced (transformed (transfected)) into host cells by known methods.
Transformation can be performed, for example, according to the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, 69:2110 (1972)), the protoplast method (Mol. Gen. Genet., 168:111 (1979)), or the competent method (J. Mol. Biol., 56:209 (1971)) when the hosts are bacteria (E. coli, Bacillus subtilis, and such), the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, 75:1927 (1978)), or the lithium method (J. Bacteriol., 153:163 (1983)) when the host is Saccharomyces cerevisiae, the method of Graham (Virology, 52:456 (1973)) when the hosts are animal cells, and the method of Summers et al. (Mol. Cell. Biol., 3:2156-2165 (1983)) when the hosts are insect cells.
The protein of the present invention can be produced by cultivating trarsformants (in the following, this term includes transfectants) comprising an expression vector prepared as mentioned above in nutrient media.
The nutrient media preferably comprises a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of host cells (transformants). Examples of the carbon source are glucose, dextran, soluble starch, and sucrose, and examples of the inorganic or organic nitrogen source are ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soy bean cake, and potato extract. If desired, they may comprise other nutrients (for example, an inorganic salt (for example, calcium chloride, sodium dihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, and so on).
Cultivation is performed by a method known in the art. Cultivation conditions such as temperature, pH of the media, and cultivation time are selected appropriately so that the protein of the present invention is produced in large quantities.
Specific media and cultivation conditions used depending on host cells are illustrated below, but are not limited thereto.
When the hosts are bacteria, actinomycetes, yeast, or filamentous fungi, liquid media comprising the nutrient source mentioned above are appropriate. Media with a pH of 5 to 8 are preferably used.
When the host is E. coli, examples of preferable media are LB media, M9 media (Miller et al. Exp. Mol. Genet., Cold Spring Harbor Laboratory, p.431 (1972)), and so on. Using these media, cultivation can be performed usually at 14 to 43xc2x0 C. for about 3 to 24 hours with aeration and stirring, if necessary.
When the host is Bacillus, cultivation can be performed usually at 30 to 40xc2x0 C. for about 16 to 96 hours with aeration and stirring, if necessary.
When the host is yeast, an example of medium is Burkholder minimal medium (Bostian, Proc. Natl. Acad. Sci. USA, 77:4505 (1980)). The pH of the medium is preferably 5 to 8. Cultivation can be performed usually at 20 to 35xc2x0 C. for about 14 to 144 hours with aeration and stirring, if necessary.
When the host is an animal cell, examples of media are MEM containing about 5 to 20% fetal bovine serum (Science, 122:501 (1952)), DMEM (Virology, 8:396 extract. Finally, the protein is isolated and purified from the crude extract by a usual method as illustrated above.
By using a DNA (cDNA or genomic DNA) encoding a human-derived AID protein included in the protein of the present invention, transgenic non-human mammals secreting the human AID protein in their body can be prepared. Namely, by integrating the human-derived DNA into an endogenous locus of non-human mammals (e.g. mouse), the human AID protein of the present invention encoded by the DNA is expressed and secreted in their body. The transgenic non-human mammals are included in the present invention.
The transgenic non-human mammals can be prepared according to the method usually used for producing a transgenic animal (for example, see xe2x80x9cNewest Manual of Animal Cell Experiment,xe2x80x9d LIC press, Chapter 7, pp.361-408, (1990)).
Specifically, for example, a transgenic mouse can be produced as follows. Embryonic stem cells (ES cells) obtained from normal mouse blastocysts are transformed with an expression vector in which the gene encoding the human AID protein of the present invention and a marker gene (for example, neomycin resistance gene) have been inserted in an expressible manner. ES cells in which the gene encoding the human AID protein of the present invention has been integrated into the endogenous gene are screened by a usual method based on expression of the marker gene. Then, the ES cells screened are microinjected into a fertilized egg (blastocyst) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, 77:7380-7384 (1980); U.S. Pat. No. 4,873,191).
The blastocyst is transplanted into the uterus of another normal mouse as the foster mother. Then, founder mice are born from the foster mother. By mating the founder mice with normal mice, heterozygous transgenic mice are obtained. By mating the heterozygous transgenic mice with each other, homozygous transgenic mice are obtained according to Mendel""s laws.
Also, a so-called xe2x80x9cknockout mousexe2x80x9d can be generated based on the nucleotide sequence of DNA encoding mouse AID protein included in the present invention. The xe2x80x9cknockout mousexe2x80x9d in the present invention means the mouse in which the endogenous gene encoding the mouse AID protein of the present invention is knocked-out (inactivated). For example, it can be generated by positive-negative selection method applying homologous recombination (U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; Proc. Natl. Acad. Sci. USA, (1959)), RPMI1640 medium (J. Am. Med. Assoc., 199:519 (1967)), 199 medium (Proc. Soc. Exp. Biol. Med., 73:1 (1950)), and so on. The pH of the medium is preferably about 6 to 8. Cultivation can be performed usually at about 30 to 40xc2x0 C. for about 15 to 72 hours with aeration and stirring, if necessary.
When the host is an insect cell, an example of medium is Grace""s medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, 82:8404 (1985)). The pH thereof is preferably about 5 to 8. Cultivation can be performed usually at about 20 to 40xc2x0 C. for 15 to 100 hours with aeration and stirring, if necessary.
The protein of the present invention can be produced by cultivating transformants, especially mammalian cells, as mentioned above and allowing them to secrete the protein into the culture supernatant.
A culture filtrate (supernatant) is obtained by a method such as filtration or centrifugation of the obtained culture, and the protein of the present invention is purified and isolated from the culture filtrate by methods commonly used in order to purify and isolate a natural or synthetic protein.
Examples of the isolation and purification method are a method utilizing solubility, such as salting out and solvent precipitation method; a method utilizing the difference in molecular weight, such as dialysis, ultrafiltration, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; a method utilizing charge, such as ion exchange chromatography and hydroxylapatite chromatography; a method utilizing specific affinity, such as affinity column chromatography; a method utilizing the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and a method utilizing the difference in isoelectric point, such as isoelectric focusing.
When the protein of the present invention exists in the periplasm or cytoplasm of cultured transformants (for example, E. coli), first, the cells are harvested by a usual method such as filtration or centrifugation and suspended in appropriate buffer. After the cell wall and/or cell membrane of the cells and such are disrupted by a method such as lysis with sonication, lysozyme, and freeze-thawing, the membrane fraction comprising the protein of the present invention is obtained by a method such as centrifugation or filtration. The membrane fraction is solubilized with a detergent such as Triton-X100 to obtain the crude 86:8932-8935, 1989; Nature, 342:435-438, 1989; etc.), and such knockout mice are one embodiment of the present invention.
The xe2x80x9cantibodyxe2x80x9d in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody, and preferably a monoclonal antibody.
Specifically, it includes an antibody reactive to the above-mentioned protein of the present invention and a fragment thereof.
The xe2x80x9cantibodyxe2x80x9d of the present invention also includes a natural antibody that can be prepared by immunizing mammals such as mice, rats, hamsters, guinea pigs, or rabbits with the protein of the present invention (including natural, recombinant, and chemically synthesized protein and cell), a fragment thereof, or a transformant highly expressing the protein of interest by recombinant technology mentioned above; a chimeric antibody and a humanized antibody (CDR-grafted antibody) that can be produced by recombinant technology; and a human monoclonal antibody that can be produced by using human antibody-producing transgenic animals.
The monoclonal antibody includes those having any one of the isotypes of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.
The polyclonal antibody (antiserum) or monoclonal antibody of the present invention can be produced by known methods. Namely, mammals, preferably, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, or cows, or more preferably, mice, rats, hamsters, guinea pigs, or rabbits are immunized, for example, with an antigen mentioned above with Freund""s adjuvant, if necessary. The polyclonal antibody can be obtained from the serum obtained from the animal so immunized. The monoclonal antibodies are produced as follows. Hybridomas are produced by fusing the antibody-producing cells obtained from the animal so immunized and myeloma cells incapable of producing autoantibodies. Then the hybridomas are cloned, and clones producing the monoclonal antibodies showing the specific affinity to the antigen used for immunizing the mammal are screened.
Specifically, the monoclonal antibody can be produced as follows. Immunizations are done by injecting or implanting once or several times the above-mentioned protein of the present invention, a fragment thereof, the cells that express the protein, and so on, as an immunogen, if necessary, with Freund""s adjuvant, subcutaneously, intramuscularly, intravenously, through the footpad, or intraperitoneally into mice, rats, hamsters, guinea pigs, or rabbits, preferably mice, rats or hamsters (including transgenic animals generated so as to produce antibodies derived from another animal such as a transgenic mouse producing human antibodies ). Usually, immunizations are performed one to four times every one to fourteen days after the first immunization. Antibody-producing cells are obtained from the mammal so immunized in about one to five days after the last immunization.
Hybridomas that secrete a monoclonal antibody can be prepared by the method of Kxc3x6hler and Milstein (Nature, 256:495-497 (1975)) and by its modified method. Namely, hybridomas are prepared by fusing antibody-producing cells contained in a spleen, lymph node, bone marrow, or tonsil obtained from the non-human mammal immunized as mentioned above, preferably a spleen, with myeloma cells without autoantibody-producing ability, which are derived from, preferably, a mammal such as mice, rats, guinea pigs, hamsters, rabbits, or humans, or more preferably, mice, rats, or humans.
For example, mouse-derived myeloma P3/X63-AG8.653 (653; ATCC No. CRL1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2), PAI, F0, or BW5147; rat-derived mycloma 210RCY3-Ag.2.3.; or human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.
Hybridoma clones producing monoclonal antibodies can be screened by cultivating the hybridomas, for example, in microtiter plates and by measuring the reactivity of the culture supernatant in the well in which hybridoma growth is observed, to the immunogen used for the immunization mentioned above, for example, by an enzyme immunoassay such as RIA and ELISA.
The monoclonal antibodies can be produced from hybridomas by cultivating the hybridomas in vitro or in vivo such as in the ascites of mice, rats, guinea pigs, hamsters, or rabbits, preferably mice or rats, more preferably mice and isolating the antibodies from the resulting culture supernatant or ascites fluid of a mammal.
In vitro cultivation can be performed depending on the property of cells to be cultured, on the object of a test study, and on various culture, by using known nutrient media or any nutrient media derived from known basal media for growing, maintaining, and storing the hybridomas to produce monoclonal antibodies in the culture supernatant.
Examples of basal media are low calcium concentration media such as Hamxe2x80x2F12 medium, MCDB153 medium, or low calcium concentration MEM medium, and high calcium concentration media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basal media can contain, for example, sera, hormones, cytokines, and/or various inorganic or organic substances depending on the objective.
Monoclonal antibodies can be isolated and purified from the culture supernatant or ascites mentioned above by saturated ammonium sulfate precipitation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), affinity chromatography using anti-immunoglobulin column or protein A column.
Furthermore, monoclonal antibodies can be obtained in a large quantity by cloning a gene encoding a monoclonal antibody from the hybridoma, generating transgenic bovines, goats, sheep, or pigs in which the gene encoding the antibody is integrated in its endogenous gene using transgenic animal generating technique, and recovering the monoclonal antibody derived from the antibody gene from milk of the transgenic animals (Nikkei Science, No.4, pp.78-84 (1997)).
The xe2x80x9cchimeric antibodyxe2x80x9d of the present invention means a monoclonal antibody prepared by genetic engineering, and specifically, a chimeric monoclonal antibody, for example, mouse/human chimeric antibody, whose variable region is a mouse immunoglobulin-derived variable region and whose constant region is a human immunoglobulin-derived constant region.
The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG, IgM, IgA, IgD, IgE, etc. The constant region of the recombinant chimeric monoclonal antibody of the present invention can be that of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG.
The chimeric monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.
For example, mouse/human chimeric monoclonal antibody can be prepared, by referring to Experimental Medicine: SUPPLEMENT, 1.6, No.10 (1988); and Examined Published Japanese Patent Application (JP-B) No. Hei 3-73280. Namely, it can be prepared by ligating CH gene (C gene encoding the constant region of H chain) obtained from the DNA encoding human immunoglobulin to the downstream of active VH genes (rearranged VDJ gene encoding the variable region of H chain) obtained from the DNA encoding mouse monoclonal antibody isolated from the hybridoma producing the mouse monoclonal antibody, and by ligating the CL gene (C gene encoding the constant region of L chain) obtained from the DNA encoding human immunoglobulin to the downstream of active VL genes (rearranged VJ gene encoding the variable region of L chain) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma, and operably inserting those into the same or different vectors in an expressible manner, followed by transformation of host cells with the expression vector, and cultivation of the transformants.
Specifically, DNAs are first extracted from mouse monoclonal antibody-producing hybridoma by the usual method, digested with appropriate restriction enzymes (for example, EcoRI and HindIII), electrophoresed (using, for example, 0.7% agarose gel), and analyzed by Southern blotting. After the electrophoresed gel is stained, for example, with ethidium bromide and photographed, the gel is given marker positions, washed twice with water, and soaked in 0.25 M HCl for 15 minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minutes with gentle stirring. The DNAs are transferred to a filter for 4 hours following the usual method. The filter is recovered and washed twice with 2xc3x97SSC. After the filter is sufficiently dried, it is baked at 75xc2x0 C. for 3 hours, treated with 0.1xc3x97SSC/0.1% SDS at 65xc2x0 C. for 30 minutes, and then soaked in 3xc3x97SSC/0.1% SDS. The filter obtained is treated with prehybridization solution in a plastic bag at 65xc2x0 C. for 3 to 4 hours.
Next, 32P-labeled probe DNA and hybridization solution are added to the bag and reacted at 65xc2x0 C. about 12 hours. After hybridization, the filter is washed under an appropriate salt concentration, reaction temperature, and time (for example, 2xc3x97SSC/0.1% SDS, room temperature, 10 minutes). The filter is put into a plastic bag with a little 2xc3x97SSC, and subjected to autoradiography after the bag is sealed.
Rearranged VDJ gene and VJ gene encoding H chain and L chain of mouse monoclonal antibody respectively are identified by Southern blotting mentioned above. The region comprising the identified DNA fragment is fractionated by sucrose density gradient centrifugation and inserted into a phage vector (for example, Charon 4A, Charon 28, xcexEMBL3, xcexEMBL4; etc.). E. coli (for example, LE392, NM539, etc.) are transformed with the phage vector to generate a genomic library. The genomic library is screened by plaque hybridization such as the Benton-Davis method (Science, 196:180-182 (1977)) using appropriate probes (H chain J gene, L chain (xcexa) J gene, etc.) to obtain positive clones comprising rearranged VDJ gene or VJ gene respectively. By making the restriction map and determining the nucleotide sequence of the clones obtained, it is confirmed that genes comprising the desired, rearranged VH (VDJ) gene or VL (VJ) gene have been obtained.
Separately, human CH gene and human CL gene used for chimerization are isolated. For example, when a chimeric antibody with human IgG1 is produced, Cxcex31 gene is isolated as a CH gene, and Cxcexa gene is also isolated as a CL gene, are isolated. These genes can be isolated from human genomic library with mouse Cxcex31 gene and mouse Cxcexa gene, corresponding to human Cxcex31 gene and human Cxcexa gene, respectively, as probes, taking advantage of the high homology between the nucleotide sequences of mouse immunoglobulin gene and that of human immunoglobulin gene.
Specifically, DNA fragments comprising human Cxcexa gene and an enhancer region are isolated from human xcex Charon 4A HaeIII-AluI genomic library (Cell, 15:1157-1174 (1978)), for example, using a 3 kb HindIII-BamHI fragment from clone Ig146 (Proc. Natl. Acad. Sci. USA, 75:4709-4713 (1978)) and a 6.8 kb EcoRI fragment from clone MEP10 (Proc. Natl. Acad. Sci. USA, 78:474-478 (1981)) as probes. In addition, for example, after human fetal hepatocyte DNA is digested with HindIII and fractioned by agarose gel electrophoresis, a 5.9 kb fragment is inserted into xcex788 and then human Cxcex31 gene is isolated with the probes mentioned above.
Using a mouse VH gene, mouse VL gene, human CH gene, and human CL gene so obtained, and taking the promoter region and enhancer region into consideration, human CH gene is inserted downstream of mouse VH gene and human CL gene is inserted downstream of mouse VL gene in an expression vector such as pSV2gpt or pSV2neo with appropriate restriction enzymes and DNA ligase following the usual method. In this case, chimeric genes of mouse VH gene/human CH gene and mouse VL gene/human CL gene can be respectively inserted into the same or a different expression vector.
Chimeric gene-inserted expression vector(s) thus prepared are introduced into myeloma cells (e.g., P3X63 Ag8 653 cells or SP210 cells) that do not produce antibodies by the protoplast fusion method, DEAE-dextran method, calcium phosphate method, or electroporation method. The transformants are screened by cultivating them in a medium containing a drug corresponding to the drug resistance gene inserted into the expression vector and, then, cells producing the desired chimeric monoclonal antibodies are obtained.
Desired chimeric monoclonal antibodies are obtained from the culture supernatant of antibody-producing cells thus screened.
The xe2x80x9chumanized antibody (CDR-grafted antibody)xe2x80x9d of the present invention is a monoclonal antibody prepared by genetic engineering and specifically means a humanized monoclonal antibody wherein a portion or the whole of the complementarity determining regions of the hyper-variable region are derived from those of the hyper-variable region from mouse monoclonal antibody, the framework regions of the variable region are derived from those of the variable region from human immunoglobulin, and the constant region is derived from that from human-immunoglobulin.
The complementarity determining regions of the hyper-variable region exists in the hyper-variable region in the variable region of an antibody and means three regions which directly bind, in a complementary manner, to an antigen (complementarity-determining residues, CDR1, CDR2, and CDR3). The framework regions of the variable region mean four comparatively conserved regions intervening upstream, downstream or between the three complementarity-determining regions (framework region, FR1, FR2, FR3, and FR4).
In other words, a humanized monoclonal antibody means that in which the whole region except a portion or the whole region of the complementarity determining regions of the hyper-variable region of a mouse monoclonal antibody has been replaced with their corresponding regions derived from human immunoglobulin.
The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG, IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonal antibody of the present invention can be that from human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. The framework regions of the constant region derived from human immunoglobulin are not particularly limited.
The humanized monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.
For example, a recombinant humanized monoclonal antibody derived from mouse monoclonal antibody can be prepared by genetic engineering, referring to Published Japanese Translations of PCT International Publication No. Hei 4-506458 and Unexamined Published Japanese Patent Application (JP-A) No. Sho 62-296890. Namely, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated from hybridomas producing mouse monoclonal antibody, and human H chain gene encoding the whole region except human H chain CDR corresponding to mouse H chain CDR mentioned above and human L chain gene encoding the whole region except human L chain CDR corresponding to mouse L chain CDR mentioned above are isolated from human immunoglobulin genes.
The mouse H chain CDR gene(s) and the human H chain gene(s) so isolated are inserted, in an expressible manner, into an appropriate vector so that they can be expressed. Similarly, the mouse L chain CDR gene(s) and the human L chain gene(s) are inserted, in an expressible manner, into another appropriate vector so that they can be expressed. Alternatively, the mouse H chain CDR gene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human L chain gene(s) can be inserted, in an expressible manner, into the same expression vector so that they can be expressed. Host cells are transformed with the expression vector thus prepared to obtain transformants producing humanized monoclonal antibody. By cultivating the transformants, desired humanized monoclonal antibody is obtained from culture supernatant.
The xe2x80x9chuman antibodyxe2x80x9d used in the present invention is immunoglobulin in which the entire regions comprising the variable and constant region of the H chain, and the variable and constant region of the L chain constituting immunoglobulin are derived from the genes encoding human immunoglobulin.
The human antibody can be produced in the same way as the production method of polyclonal or monoclonal antibodies mentioned above by immunizing, with an antigen, a transgenic animal which for example, at least human immunoglobulin gene(s) have been integrated into the locus of a non-human mammal such as a mouse by the usual method.
For example, a transgenic mouse producing human antibodies is prepared by the methods described in already published literatures (Nature Genetics, 7:13-21 (1994); Nature Genetics, 15:146-156 (1997); JP-WA Hei 4-504365; WO94/25585; Nikkei Science, No.6, pp.40-50 (1995); WO94/25585; Nature, 368:856-859 (1994); JP-WA No. Hei 6-500233).
The xe2x80x9cportion of an antibodyxe2x80x9d used in the present invention means a partial region of the antibody, and preferably the monoclonal antibody of the present invention as mentioned above, and specifically, means F(abxe2x80x2)2, Fabxe2x80x2, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domain antibody) (Exp. Opin. Ther. Patents, 6, No.5, pp.441-456 (1996)).
xe2x80x9cF(abxe2x80x2)2xe2x80x9d and xe2x80x9cFabxe2x80x2xe2x80x9d can be produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and means an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate two homologous antibody fragments in which an L chain composed of VL (L chain variable region) and CL (L chain constant region), and an H chain fragment composed of VH (H chain variable region) and CHxcex31 (xcex31 region in the constant region of H chain) are connected at their C terminal regions through a disulfide bond. Each of these two homologous antibody fragments is called Fabxe2x80x2. Pepsin also cleaves IgG downstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned Fabxe2x80x2 are connected at the hinge region. This antibody fragment is called F(abxe2x80x2)2.
The xe2x80x9ccell producing a monoclonal antibody reactive to a protein or a fragment thereofxe2x80x9d of the present invention means any cell producing the above-described monoclonal antibody of the present invention.
More specifically, the following is included:
(1) B cells that are obtained by immunizing the non-human mammals with the above-mentioned protein of the present invention, a fragment thereof, or the cells producing the protein and that produce a monoclonal antibody reactive to the protein of the present invention or a fragment thereof.
(2) The above-mentioned hybridomas (fused cell) prepared by fusing the thus-obtained B cells producing the antibody with myeloma cells derived from mammals.
(3) Monoclonal antibody-producing transformants obtained by transforming cells other than the monoclonal antibody-producing B cells and hybridomas with genes encoding the monoclonal antibody isolated from the monoclonal antibody-producing B cells or hybridomas (either the heavy chain-encoding gene or the light chain-encoding gene, or both).
The monoclonal antibody-producing transformants of (3) mean recombinant cells producing a recombinant monoclonal antibody produced by B cells of (1) or hybridomas of (2). These antibody producing-transformants can be produced by the method as used for producing the above-described chimeric monoclonal antibody and humanized monoclonal antibody.
The xe2x80x9cpharmaceutical compositionxe2x80x9d used herein means a pharmaceutical composition comprising of any of the protein, fragment thereof, antibody, or portion thereof defined hereinabove, and a pharmaceutically acceptable carrier.
The xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d includes an excipient, a diluent, an expander, a disintegrating agent, a stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a colorant, a sweetener, a viscosity-increasing agent, a flavor, a dissolving agent, or other additives. Using one or more of such carriers, a pharmaceutical composition can be formulated into tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions, or syrups. The pharmaceutical composition can be administered orally or parenterally. Other forms for parenteral administration include a solution for external application, suppository for rectal administration, and pessary, prescribed by the usual method, which comprises one or more active ingredient.
The dosage can vary depending on the age, sex, weight, and symptoms of a patient, effect of treatment, administration route, period of treatment, or the kind of active ingredient (protein or antibody mentioned above) contained in the pharmaceutical composition. Usually, the pharmaceutical composition can be administered to an adult in a dose of 10 xcexcg to 1000 mg (or 10 xcexcg to 500 mg) per one administration. Depending on various conditions, the lower dosage may be sufficient in some cases, and a higher dosage may be necessary in other cases.
In particular, the injection can be produced by dissolving or suspending the antibody in a non-toxic, pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injections by adjusting the concentration to 0.1 xcexcg antibody/ml carrier to 10 mg antibody/ml carrier. The injection thus produced can be administered to a human patient in need of treatment in a dose of 1 xcexcg to 100 mg/kg body weight, preferably 50 xcexcg to 50 mg/kg body weight, once or more times a day. Examples of administration routes are medically appropriate administration routes such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection, preferably intravenous injection.
The injection can also be prepared into a non-aqueous diluent (for example, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and alcohols such as ethanol), suspension, or emulsion.
The injection can be sterilized by filtration with a bacteria-non-penetrable filter, by mixing bacteriocide, or by irradiation. The injection can be prepared at the time of use. Namely, it is freeze-dried to make a sterile solid composition, and can be dissolved in sterile distilled water for injection or another solvent before use.
The pharmaceutical composition of the present invention is useful as a drug for preventing and treating, for example, primary immunodeficiency syndrome with congenital disorder of immune system, mainly immunodeficiency considered to develop by B lymphocyte deficiency, decrease, or dysfunction (e.g., sex-linked agammaglobulinemia, sex-linked agammaglobulinemia with growth hormone deficiency, immunoglobulin deficiency with high IgM level, selective IgM deficiency, selective IgE deficiency, immunoglobulin heavy chain gene deletion, xcexa chain deficiency, IgA deficiency, IgG subclass selective deficiency, CVID (common variable immunodeficiency), infantile transient dysgammaglobulinemia, Rosen syndrome, severe combined immunodeficiency (sex-linked, autosomal recessive), ADA (adenosine deaminase) deficiency, PNP (purine nucleoside phosphorylase) deficiency, MHC class II deficiency, reticular dysplasia, Wiskott-Aldrich syndrome, ataxia telangiectasia, DiGeorge syndrome, chromosomal aberration, familial Ig hypermetabolism, hyper IgE syndrome, Gitlin syndrome, Nezelof syndrome, Good syndrome, osteodystrophy, transcobalamin syndrome, secretory bead syndrome, etc.), various diseases with antibody production deficiency that are secondary immunodeficiency syndrome with disorder of immune system caused by an acquired etiology (for example, AIDS, etc.), and/or various allergic diseases (e.g., bronchial asthma, atopic dermatitis, conjunctivitis, allergic rhinitis, allergic enteritis, drug-induced allergy, food allergy, allergic urticaria, glomerulonephritis, etc.), and for relieving conditions due to various immunodeficiencies associated with the diseases.
The DNA of the present invention described above, namely, xe2x80x9cDNA comprising any partial nucleotide sequence of SEQ ID NO:7, from SEQ ID NO:9 to SEQ ID NO:15, or SEQ ID NO:35, those with partial chemical modification, DNA comprising complementary nucleotide sequences to the partial sequence, or those with partial chemical modificationxe2x80x9d are included.
Here, the xe2x80x9cpartial nucleotide sequencexe2x80x9d means the partial nucleotide sequence comprising any number of bases at any region included in any nucleotide sequence listed in SEQ ID NO:7, from SEQ ID NO:9 to SEQ ID NO:15, or SEQ ID NO:35.
The DNA is useful as probes in DNA hybridization or RNA hybridization procedures. For the purpose of using the DNA as a probe, continuous nucleotide sequences of over 20 bases, preferably continuous nucleotide sequences of over 50 bases, more preferably over 100 bases, much more preferably over 200 bases, especially preferably over 300 bases, can be used as the partial nucleotide sequences.
Also, the DNA described above, as mentioned before, are useful as primers for PCR. For the purpose of using the DNA as PCR primers, continuous partial nucleotide sequences of from 5 to 100 bases, preferably from 5 to 70 bases, more preferably from 5 to 50 bases, much more preferably from 5 to 30 bases, can be used as the partial nucleotide sequences.
Moreover, the DNA described above are useful as antisense drug. The DNA, by hybridizing to a DNA or an RNA encoding the AID protein of the present invention, can inhibit transcription of the DNA to mRNA or translation of the mRNA into the protein.
For the purpose of using above-mentioned DNA to antisense drug, the partial nucleotide sequence consists of 5 to 100 consecutive nucleotides, preferably 5 to 70 consecutive nucleotides, more preferably 5 to 50 consecutive nucleotides, and still more preferably 5 to 30 consecutive nucleotides.
When the DNA is used as an antisense DNA pharmaceutical, the DNA sequence can be modified chemically in part for extending the half-life (stability) of the blood concentration of the DNA administered to patients, for increasing the intracellular-membrane permeability of the DNA, or for increasing the degradation resistance or the absorption of the orally administered DNA in the digestive organs. The chemical modification includes, for example, the modification of the phosphate bonds, the riboses, the nucleotide bases, the sugar moiety, the 3xe2x80x2 end and/or the 5xe2x80x2 end in the structure of the oligonucleotide DNA.
The modification of phosphate bonds includes, for example, the conversion of one or more of the bonds to phosphodiester bonds (D-oligo), phosphorothioate bonds, phosphorodithioate bonds (S-oligo), methyl phosphonate (MP-oligo), phosphoroamidate bonds, non-phosphate bonds or methyl phosphonothioate bonds, or combinations thereof. The modification of the ribose includes, for example, the conversion to 2xe2x80x2-fluororibose or 2xe2x80x2-O-methylribose. The modification of the nucleotide base includes, for example, the conversion to 5-propynyluracil or 2-aminoadenine.
Also, another embodiment of the present invention relates to xe2x80x9cmethods of identifying substances regulating the production of the AID protein of the present invention or the transcription of the gene encoding AID protein to mRNA.xe2x80x9d The method of the present invention is namely xe2x80x9cthe method of screening of drugs capable of regulating functions of AID protein or AID gene.xe2x80x9d
As the cells in the method of the present invention, any cells, as long as capable of producing AID protein of the present invention, can be used. For instance, native cells (preferably of mouse or human), transgenic cells transformed with a gene encoding an AID protein of the present invention, cells introduced with RNA encoding an AID protein of the present invention, etc., can be used.
As the host cells for preparing the transgenic cells, various cells, mentioned in the part explaining in detail the methods of expressing the protein of the present invention using the DNA of the protein described above, can be used.
For instance, various cells such as naturally established cells or artificially established transgenic cells (e.g. bacteria (Escherichia, Bacillus), yeast (Saccharomyces, Pichia), animal cells and insect cells) can be exemplified.
Preferably, animal cells, namely, cells derived from mouse (COP, L, C127, Sp2/0, NS-1, or NIH3T3, etc.), cells derived from rat (PC12, PC12h, etc.), cells derived from hamster (BHK, and CHO, etc.), cells derived from monkey (COS1, COS3, COS7, CV1, and Velo, etc.), and cells derived from human (Hela, cells derived from diploid fibroblast, HEK293 cells, myeloma cells, and Namalwa, etc.) can be exemplified.
The xe2x80x9csubstancexe2x80x9d in the present invention means natural substance existing in the nature and any substance prepared artificially. The substances can be grouped into xe2x80x9cpeptidic substancexe2x80x9d and xe2x80x9cnon-peptidic substance.xe2x80x9d
As the xe2x80x9cnon-peptidic substance,xe2x80x9d xe2x80x9cDNA comprising partial nucleotide sequence, or chemically modified DNA derived from itxe2x80x9d that are useful as antisense drugs as described above, xe2x80x9cantisense RNAxe2x80x9d with similar structural and pharmacological property to the antisense DNA, or any chemically synthesized xe2x80x9ccompoundsxe2x80x9d can be exemplified. Examples of xe2x80x9ccompoundsxe2x80x9d are compounds other than DNA, RNA, and the above-mentioned peptidic substances, which have a molecular weight from approximately 100 to approximately 1000, preferably from approximately 100 to approximately 800, more preferably from approximately 100 to approximately 600.
As the xe2x80x9cpeptidic substance,xe2x80x9d antibodies already described above in detail (preferably monoclonal antibodies, more preferably recombinant antibodies or human monoclonal antibodies), oligopeptides, or chemically modified substance derived from them can be exemplified. Examples of an oligopeptide are a peptide comprising 5 to 30 amino acids, preferably 5 to 20 amino acids. The chemical modification can be designed depending on various purposes, for example, for increased half-life in blood in the case of administering in vivo, or for increased tolerance against degradation or increased absorption in digestive tract after oral administration.
Methods described in from (24) to (28) above include so-called reporter gene assays, as one of the method of the present invention.
As the xe2x80x9creporter protein,xe2x80x9d luciferase derived from firefly or sea pansy, or GFP derived from jellyfish, are preferred.
As the xe2x80x9creporter gene assay,xe2x80x9d methods described below are representative.
Transgenic cells are generated by transforming cells commonly used in the production of recombinant proteins with expression vector, in which a gene encoding the target protein and a gene encoding the reporter protein are inserted into the vector so that the transcription of the gene encoding the reporter protein to mRNA is induced by the signal of the transcription of the gene of target protein to mRNA. The test substances (described above) are applied to the obtained transformant cells. Analysis that whether the compound affects the expression of transporter molecule can be accomplished by measuring the level of the target protein by indirect measurement of the amount of fluorescence emitted by the reporter protein expressed in parallel with the target protein (for reference, see U.S. Pat. No. 5,436,128 and U.S. Pat. No. 5,401,629).
The identification of the compounds using the present assay can be accomplished by manual operation, but it can also be readily and simply done automatically by so-called High-Throughput Screening using robots (SOSHIKI BAIYO KOUGAKU, 23:521-524; U.S. Pat. No. 5,670,113).
The terms xe2x80x9ccellsxe2x80x9d and xe2x80x9csubstancesxe2x80x9d used in the methods described above have the same meaning as defined above.
The substances identified by the methods of the present invention are very useful as drugs for the therapy of various diseases considered to be caused by the hyperfunction or deficiency of the AID protein of the present invention or by the deficiency or mutation of the AID gene, or for remission of various symptoms associated with the diseases.